Environmental response type charge conversion carbon quantum dot as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of carbon quantum dot materials, and particularly relates to an environmental response type charge conversion carbon quantum dot as well as a preparation method and application thereof, and the preparation method of the quantum dot comprises the following steps: (1) preparing chlorogenic acid into quantum dots to obtain ChACDs; and (2) modifying the ChA CDs by using an imidazole compound to obtain the environmental response type charge conversion carbon quantum dot. The quantum dot has excellent biocompatibility and antibacterial activity, and also has remarkable capabilities of treating skin infection and promoting wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of carbon quantum dot materials, and in particular relates to an environmentally responsive charge conversion carbon quantum dot and a preparation method and application thereof. Background Art

[0002] Antibiotic resistance is one of the threats to human health. When antibiotics act on bacteria, certain genes in the bacterial genome mutate, resulting in reduced or complete loss of bacterial sensitivity to antibiotics. These mutations may be spontaneous or caused by the selective pressure of antibiotics. In particular, antibiotic resistance caused by the selective pressure of antibiotics poses a great threat to health (it takes 4-5 years for bacteria to develop resistance through their own gene mutations, while it only takes 2-3 months for antibiotic resistance to spread in biofilms). Specifically, bacteria generally exist in two survival states: planktonic state and biofilm colonization state. During the infection process, bacteria wrap bacterial communities by secreting extracellular polymeric substances (EPSs) to form a dense three-dimensional structure called biofilms, which serves as a protective barrier for bacteria to resist the host immune system, environmental pressure and antibiotics. Although antibiotics can effectively kill bacteria in the planktonic state and the bacterial community on the surface of the biofilm, bacteria colonized in the deep layer can often avoid the effects of antibiotics and continue to survive due to the unique dense structure of the biofilm. If bacterial biofilms exist in the patient's body for a long time, they will continuously release planktonic bacteria to cause new infections, leading to chronic and persistent infections. In addition, mature biofilms can be hundreds to thousands of times more resistant to most antibiotics than single planktonic bacteria, greatly increasing the difficulty of clinical treatment. Therefore, effective treatment strategies to combat already formed bacterial biofilms are of great significance.

[0003] In order to eradicate biofilm, a series of nanomaterials have been designed and used in in vitro and in vivo experiments to confirm their effectiveness, including organic nanoparticles (such as liposomes, polymer nanoparticles, dendrimers, cyclodextrins, solid lipid nanoparticles, etc.) and inorganic nanoparticles (such as metal nanoparticles such as gold, silver, and copper, metal oxide nanoparticles such as zinc oxide and aluminum oxide, quantum dots, fullerenes, and organic-inorganic hybrid materials, etc.). Although metals such as gold and silver and metal oxide nanoparticles such as ferrous oxide, copper oxide, and zinc oxide show strong antibacterial activity, the release of metal ions caused by these materials in vivo can produce nonspecific biological toxicity, which greatly limits the application of nanomaterials in the antibacterial field.

[0004] Carbon dots (CDs) are gaining more and more attention due to their advantages such as economical and simple preparation, high water solubility, high degree of functionalization and good biocompatibility. In addition, their small size allows them to pass through various natural biological barriers in the body, including ion channels, blood-brain barrier and glomerular filtration barrier. These characteristics of CDs are expected to break the application limitations of nanomaterials in the antibacterial field and show great potential in the biomedical field. Summary of the invention

[0005] Based on this, the present invention provides an environmentally responsive charge-conversion carbon quantum dot, which is prepared by chlorogenic acid and then modified with an imidazole compound; it can selectively protonate the imidazole group in an acidic environment to achieve charge conversion, can quickly damage and penetrate the biofilm and attach to the bacteria colonized in the biofilm, thereby achieving the purpose of resisting drug-resistant bacteria.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] The present invention provides a method for preparing environmentally responsive charge conversion carbon quantum dots, comprising: (1) preparing chlorogenic acid into quantum dots to obtain ChA CDs; and (2) modifying ChA CDs with imidazole compounds to obtain environmentally responsive charge conversion carbon quantum dots.

[0008] Preferably, the above imidazole compound is n is an integer and 1≤n≤5.

[0009] More preferably, the imidazole compound is 1-(3-aminopropyl)imidazole.

[0010] Preferably, the mass / volume of ChACDs and imidazole compounds is (3-6) mg / mL.

[0011] Preferably, the preparation method satisfies one or more combinations of the following conditions: (a) in step (1), chlorogenic acid is prepared to obtain ChACDs by a hydrothermal synthesis method; (b) in step (2), the imidazole compound is modified to ChACDs by an EDC / NHS method.

[0012] More preferably, in the above preparation method, step (1) comprises: placing the ChA aqueous solution in a high-pressure reactor, and then heating it at 220°C-240°C to obtain a ChACDs aqueous solution; step (2) comprises: (i) mixing EDC, NHS and 2-morpholineethanesulfonic acid to obtain a reactant; (ii) mixing the reactant with an imidazole compound, adjusting the pH to 7-9, and reacting to obtain environmentally responsive charge conversion carbon quantum dots.

[0013] Another aspect of the present invention provides an environmentally responsive charge conversion carbon quantum dot prepared by the preparation method of the present invention.

[0014] In another aspect, the present invention provides any one of the following substances:

[0015] (i) A composition comprising the environmentally responsive charge conversion carbon quantum dots of the present invention; the composition is used for antibacterial, inhibiting the growth of drug-resistant bacteria, treating skin suppurative infections or promoting wound healing;

[0016] (ii) a formulation comprising the environmentally responsive charge conversion carbon quantum dots of the present invention or the composition of (i); and a carrier.

[0017] Another aspect of the present invention provides an application of the environmentally responsive charge conversion carbon quantum dots in the present invention in antibacterial applications.

[0018] Preferably, the above-mentioned application includes: application of the environmentally responsive charge conversion carbon quantum dots of the present invention in antibiotic-resistant bacteria.

[0019] More preferably, the antibiotic-resistant bacteria include one or more of methicillin-resistant Staphylococcus aureus, penicillin-resistant Enterococci, vancomycin-resistant Enterococci, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, or pan-resistant Acinetobacter.

[0020] Preferably, the above-mentioned application includes: application of the environmentally responsive charge conversion carbon quantum dots of the present invention in the preparation of a composition or preparation, the composition or preparation being used to destroy the bacterial membrane system, reduce the bacterial metabolism level or promote the accumulation of bacterial ROS.

[0021] Preferably, the above-mentioned application includes: application of the environmentally responsive charge conversion carbon quantum dots of the present invention in the preparation of a pharmaceutical composition or a pharmaceutical preparation, and the pharmaceutical composition or the pharmaceutical preparation is used to treat suppurative skin infection and / or promote wound healing.

[0022] The beneficial effects of the present invention include at least:

[0023] (1) The environmentally responsive charge conversion carbon quantum dots provided in the present invention have excellent biocompatibility, and toxicity tests show that they have no obvious toxic side effects and have significant antibacterial activity.

[0024] (2) The environmentally responsive charge-converting carbon quantum dots provided in the present invention have excellent antibacterial activity against antibiotic-resistant bacteria, for example, the antibacterial rate against MRSA (methicillin-resistant Staphylococcus aureus) and PAE (penicillin-resistant Enterococcus) can reach 99%.

[0025] (3) The environmentally responsive charge-converting carbon quantum dots provided in the present invention have significant abilities to treat skin infections and promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 1 is the physicochemical characterization of ChACDs and CDs-API; a is the transmission electron microscopy image and dynamic light scattering (DLS) particle size distribution diagram of ChACDs; b is the transmission electron microscopy image and DLS particle size distribution diagram of CDs-API (inset: high-resolution transmission electron microscopy image); c is the fluorescence emission spectra of ChACDs and CDs-API at 400nm to 600nm under 370nm excitation light (inset: the color of aqueous solutions of ChACDs (left) and CDs-API (right) under sunlight and ultraviolet light);

[0027] Figure 2 Structural characterization of ChACDs and CDs-API, where a is the full element scanning XPS spectrum of ChACDs; b is the C element scanning XPS spectrum of ChACDs; c is the full element scanning XPS spectrum of CDs-API; d is the Fourier transform infrared (FTIR) spectrum of ChA CDs and CDs-API;

[0028] Figure 3 Zeta potential of ChACDs and CDs-API under different pH environments;

[0029] Figure 4 The glutathionease-like activity of CDs-API is verified; a is a schematic diagram of the principle of DTNB colorimetric method for detecting GSH; b is that the catalytic activity is concentration-dependent and time-dependent;

[0030] Figure 5 Evaluation of the in vitro antibacterial properties of CDs-API; a is the plate colony map after different treatments; b is the statistics of bacterial growth in liquid culture medium after MRSA treatment; c is the statistics of bacterial growth in liquid culture medium after PAE treatment; (**p<0.01, ***p<0.001);

[0031] Figure 6 MIC evaluation of CDs-API: growth of MRSA and PAE on agar plates and liquid culture medium respectively under treatment with different concentrations of CDs-API; a is the growth of MRSA and PAE on agar plates respectively under treatment with different concentrations of CDs-API; b is the growth of MRSA in liquid culture medium under treatment with different concentrations of CDs-API; c is the growth of PAE in liquid culture medium under treatment with different concentrations of CDs-API; (*p<0.05, **p<0.01, ***p<0.001);

[0032] Figure 7 The growth of Escherichia coli, Klebsiella pneumoniae, and extended-spectrum β-lactamase-producing Klebsiella pneumoniae on LB agar plates under different treatments;

[0033] Figure 8 The crystal violet method was used to evaluate the ability of CDs-API to destroy mature biofilms of MRSA (a) and PAE (b) (**p<0.01);

[0034] Fig. 9 SEM images of MRSA and PAE after treatment with CDs-API and control PBS (scale bar: 1 μm);

[0035] Fig.10 is the PI fluorescence value of MRSA and PAE after treatment with CDs-API and control PBS;

[0036] Fig.11 is the diSC3(5) fluorescence value of MRSA and PAE after treatment with CDs-API and control PBS;

[0037] Fig.12 ATP levels of MRSA and PAE bacterial suspension after different treatments; (***p<0.001);

[0038] Fig.13 The absorbance of ABDA at 378 nm after MRSA and PAE were treated with ChACDs and CDs-API at different times;

[0039] Fig.14 MTT toxicity evaluation of HUVEC after treatment with a series of concentrations of CDs-API;

[0040] Fig.15 The weight changes of mice during the treatment period; a is the MRSA infection group, and b is the PAE infection group;

[0041] Fig.16 H&E staining of the main organs (heart, liver, spleen, lung, and kidney) of mice in each group; a is the MRSA infection group; b is the PAE infection group; (scale bar: 100 μm);

[0042] Fig.17 This is the timeline of the mouse skin purulent infection experiment;

[0043] Fig.18 The in vivo antibacterial properties of CDs-API treated with MRSA; a is the changes in the infected site of mice during treatment; b is the bacterial growth in the infected site of mice after treatment in different treatment groups; c is the H&E staining of the skin at the infected site after treatment in different treatment groups; (scale bar: 100 μm);

[0044] Fig.19 The in vivo antibacterial properties of PAE-treated CDs-API; a is the changes in the infection site of mice during the treatment; b is the bacterial growth in the infection site of mice after treatment in different treatment groups; c is the H&E staining of the skin at the infection site after treatment in different treatment groups; (Scale bar: 100 μm). DETAILED DESCRIPTION

[0045] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0046] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, materials, or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, materials, or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0047] The present invention provides a method for preparing environmentally responsive charge conversion carbon quantum dots, comprising: (1) preparing chlorogenic acid (ChA) into quantum dots to obtain ChACDs; (2) modifying ChACDs with imidazole compounds to obtain environmentally responsive charge conversion carbon quantum dots (abbreviated as CDs-API, the same below).

[0048] It should be noted that the present invention achieves acidic response charge conversion by modifying the surface of ChACDs with imidazole groups and selectively protonating the imidazole groups in a low pH environment, that is, the quantum dots can selectively protonate the imidazole groups in an acidic environment to achieve charge conversion, and can quickly penetrate the biofilm and attach to the bacteria colonized in the biofilm. The enzyme-like activity of CDs-API can effectively consume the high level of reduced glutathione in the biofilm, inducing the accumulation of reactive oxygen species (ROS), degrading the matrix of extracellular polymers, destroying bacterial DNA and protein structures, and destroying the redox balance of bacteria to kill bacteria.

[0049] It should also be noted that chlorogenic acid (ChA), also known as coffee tannic acid or coffee tannic acid, is a naturally occurring polyphenolic compound, mainly formed by ester condensation of caffeic acid and quinic acid. It is found in high concentrations in honeysuckle, eucommia bark, and coffee beans, and is known as "plant gold". It has multiple biological activities such as antiviral, antibacterial, and free radical scavenging. ChACDs are highly soluble in water and have significant glutathionease-like activity. They break the redox balance of cells by consuming GSH, and induce cell oxidative stress and death. This property provides the possibility for the application of CDs in the field of antibacterial. However, this low-toxicity CDs maintains a negative charge in aqueous solution. Compared with positively charged nanomaterials, although it avoids damage to normal tissue cells to a certain extent and has higher biocompatibility, its antibacterial effect is also inhibited to a certain extent. Therefore, the present invention further uses imidazole compounds to modify the surface of ChACDs to obtain environmentally responsive charge conversion carbon quantum dots to improve their antibacterial effect.

[0050] It should also be noted that the environmentally responsive charge conversion carbon quantum dots (CDs-API) in the present invention maintain a negative charge under normal physiological conditions (pH = 5.5), reducing nonspecific effects on normal cells and tissues, and accumulating at positively charged infection sites through electrostatic attraction; after contacting the acidic environment of the biofilm, the imidazole group of the surface functional group is protonated, quickly reversing the surface charge of the CDs-API to positive, and the CDs-API gradually adsorbs on the negatively charged bacteria to exert the material's enzyme-like activity and antibacterial activity to kill the fixed bacteria in the biofilm and destroy the biofilm. The present invention has verified that CDs-API has excellent bactericidal activity, can effectively inhibit the growth of drug-resistant bacteria, and also has a certain destructive effect on mature biofilms.

[0051] In some specific examples, the imidazole compound can be n is an integer and 1≤n≤5.

[0052] It should be noted that the imidazole compound in the present invention can be preferably The compound shown, wherein n can be 1, 2, 3, 4 or 5; wherein n is more preferably 3, that is, the imidazole compound is 1-(3-aminopropyl)imidazole, and the environmentally responsive charge conversion carbon quantum dots prepared by the imidazole compound have the best performance.

[0053] In some specific examples, the mass / volume of ChACDs and imidazole compounds is (3-6) mg / mL.

[0054] It should be noted that the imidazole compound (1-(3-aminopropyl)imidazole) is in liquid form at room temperature, so "mass / volume" refers to the ratio of the mass of ChACDs to the volume of the imidazole compound; the ratio is (3-6) mg / mL, such as 3 mg / mL, 4 mg / mL or 5 mg / mL; in addition, it should be understood that "mg / mL" can be uniformly converted according to international standard units, and does not specifically include only "mg / mL" here.

[0055] In some specific examples, the above preparation method satisfies one or more combinations of the following conditions:

[0056] (a) In step (1), chlorogenic acid (ChA) is prepared by a hydrothermal synthesis method to obtain ChACDs; specifically, the present invention uses ChA as a precursor substance and can synthesize ChACDs by a traditional "top-down" method - a hydrothermal method; it should be understood that the hydrothermal method in the present invention is carried out in a manner known in the art;

[0057] (b) In step (2), the imidazole compound is modified to ChA CDs by the EDC / NHS method; specifically, in the present invention, the imidazole compound can be modified to ChA CDs by various methods, such as the EDC / NHS method; wherein the EDC / NHS method is carried out in a manner known in the art.

[0058] In some specific examples, in the above preparation method,

[0059] Step (1) comprises: placing a ChA aqueous solution in a high pressure reactor, and then heating it at 220°C-240°C to obtain a ChACDs aqueous solution; specifically, the method of synthesizing ChACDs by hydrothermal method in the present invention can be obtained by heating the high pressure reactor in an oven, and the temperature of the oven can be 220°C-240°C, such as 225°C, 230°C or 235°C;

[0060] Step (2) comprises: (i) mixing EDC, NHS and 2-morpholineethanesulfonic acid to obtain a reactant; (ii) mixing the reactant with an imidazole compound, adjusting the pH to 7-9, and reacting to obtain environmentally responsive charge conversion carbon quantum dots.

[0061] The embodiment of the present invention also provides an environmentally responsive charge conversion carbon quantum dot prepared by the preparation method of the present invention.

[0062] The present invention also provides any one of the following substances:

[0063] (i) A composition comprising the environmentally responsive charge conversion carbon quantum dots of the present invention; the composition is used for antibacterial, inhibiting the growth of drug-resistant bacteria, treating skin suppurative infections or promoting wound healing;

[0064] (ii) a formulation comprising the environmentally responsive charge conversion carbon quantum dots of the present invention or the composition of (i); and a carrier.

[0065] It should be noted that the above-mentioned composition refers to the environmentally responsive charge-conversion carbon quantum dots in the present invention prepared in combination with other effective active compounds; for example, the antibacterial composition may be composed of environmentally responsive charge-conversion carbon quantum dots and one or more compounds (such as antibiotics) with antibacterial activity; for example, the composition used to inhibit the growth of drug-resistant bacteria may be composed of environmentally responsive charge-conversion carbon quantum dots and compounds with the activity of inhibiting the growth of drug-resistant bacteria; similarly, the composition of drugs for treating purulent skin infections or the composition of drugs for promoting wound healing is composed of environmentally responsive charge-conversion carbon quantum dots and a drug composition that can treat purulent skin infections or a compound that promotes wound healing; in addition, it should be understood that the purpose of the composition of the compound can be to enhance the therapeutic effect or to combine different therapeutic effects, and the specific selection can be based on clinical practice.

[0066] It should also be noted that the preparation can be the environmentally responsive charge conversion carbon quantum dots in the present invention or the above-mentioned composition added with a carrier to prepare different preparations, such as sprays, injections, powders, tablets, capsules, ointments or patches, etc.; of course, it should be understood that the choice of carrier can be based on different dosage forms according to the types known in the art; in addition, in specific products, antibacterial preparations can be in the form of sprays, ointments or patches, etc.; pharmaceutical preparations for inhibiting the growth of antibiotic-resistant bacteria can be tablets, powders or capsules, etc.; pharmaceutical preparations for treating purulent skin infections or promoting wound healing can be powders, capsules, injections or patches, etc.

[0067] The embodiment of the present invention also provides an application of the environment-responsive charge-converting carbon quantum dots in the present invention in antibacterial applications.

[0068] In some specific examples, the above applications include: application of the environmentally responsive charge-converting carbon quantum dots of the present invention in antibiotic-resistant bacteria.

[0069] In some specific examples, the antibiotic-resistant bacteria include one or more of methicillin-resistant Staphylococcus aureus, penicillin-resistant Enterococci, vancomycin-resistant Enterococci, carbapenem-resistant Enterobacter, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, or pan-resistant Acinetobacter.

[0070] It should be noted that the antibiotic-resistant bacteria in the present invention are well known to the present invention, such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, carbapenem-resistant Enterobacter, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa or pan-resistant Acinetobacter (usually resistant to antibiotics such as aminoglycosides, cephalosporins, penicillins, tetracyclines, sulfonamides, etc.) and the like.

[0071] In some specific examples, the above applications include: the use of the environmentally responsive charge-conversion carbon quantum dots in the present invention in the preparation of compositions or preparations, which are used to destroy bacterial membrane systems, reduce bacterial metabolic levels, or promote bacterial ROS accumulation.

[0072] It should be noted that when evaluating the antibacterial properties of the materials in vitro, it was observed that CDs-API showed a more significant antibacterial effect than ChACDs; however, the two materials showed similar ROS generation capabilities. The antibacterial mechanism of CDs-API may include the following aspects: (1) Cell membrane damage: CDs-API can damage the bacterial cell membrane, causing changes in membrane permeability and membrane potential; (2) Decreased metabolic level: CDs-API reduces the metabolic level of bacteria, which may be an important factor leading to bacterial death; (3) GSH consumption and ROS accumulation: CDs-API can exert GSH-like enzyme activity, consume reduced GSH in bacteria, lead to ROS accumulation, and then damage the DNA and protein structure of bacteria in the extracellular matrix.

[0073] In some specific examples, the above-mentioned applications include: the use of the environmentally responsive charge-conversion carbon quantum dots in the present invention in the preparation of pharmaceutical compositions or pharmaceutical preparations, and the pharmaceutical compositions or pharmaceutical preparations are used to treat suppurative skin infections and / or promote wound healing.

[0074] It should be noted that, in the present invention, it is proved through animal experiments that CDs-API can effectively treat suppurative skin infections in mice and has a certain promoting effect on wound healing; CDs-API has low cytotoxicity and good biocompatibility in mice.

[0075] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0076] 1. Preparation of Quantum Dot CDs-API

[0077] Example 1

[0078] (1) Weigh 150 mg ChA (chlorogenic acid) into a 50 mL centrifuge tube, add 30 mL ultrapure water, and transfer to a 50 mL polytetrafluoroethylene-lined autoclave after fully dissolving. Preheat the high-temperature oven to 230 ° C in advance. After the temperature stabilizes, put the autoclave containing the ChA aqueous solution into the oven and heat for 2 h. Take out the autoclave and cool it naturally to room temperature, then take out the liner, filter the solution through a microporous filter membrane (0.02 μm) to remove impurities, and put the obtained brown transparent solution into a dialysis bag (1 kDa). Dialyze and purify it with ultrapure water for 8 h to obtain a ChACDs aqueous solution (the solution can be freeze-dried according to experimental requirements);

[0079] (2) Weigh 50 mg of EDC (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride) and 25 mg of NHS (N-hydroxysuccinimide) was placed in a 10 mL centrifuge tube, and 1 mL MES (2-morpholineethanesulfonic acid) (pH = 5.5) was added to fully dissolve it; in a light-proof environment, 2 mL of the above-synthesized ChACDs aqueous solution was added to a centrifuge tube and incubated at 37°C for 1 hour; immediately thereafter, 1 mL API (1-(3-aminopropyl) imidazole) was added, and the pH of the solution was adjusted to 8 with PBS, and the reaction solution was incubated at 4°C overnight; the reacted solution was transferred to a dialysis bag (molecular cutoff 3 kDa), and then dialyzed and purified with ultrapure water for 24 hours (in this process, ultrapure water was replaced every 2 hours to ensure that small molecule impurities were completely removed); after the dialysis was completed, the light brown solution was taken out from the dialysis bag and freeze-dried to obtain a stable solid light yellow powder, which was the product CDs-API-1 (environmentally responsive charge conversion carbon quantum dots), and stored at 4°C in the dark for later use.

[0080] Example 2

[0081] The difference between Example 2 and Example 1 is that the imidazole compound is different, and the imidazole compound in Example 2 is 1-(3-aminoethyl)imidazole; the other steps are the same as Example 1, and quantum dots CDs-API-2 are prepared.

[0082] Example 3

[0083] The difference between Example 3 and Example 1 is that the imidazole compound is different, and the imidazole compound in Example 3 is 1-(3-aminobutyl)imidazole; the other steps are the same as Example 1, and quantum dots CDs-API-3 are prepared.

[0084] Comparative Example 1

[0085] The difference between Comparative Example 1 and Example 1 is that the carbon dot precursor is different. The carbon dot precursor used in Comparative Example 1 is urea citrate. The other steps are the same as those in Example to prepare quantum dots CDs-API-4.

[0086] 2. Quantum dot CDs-API characterization

[0087] In the following experiments, CDs-API refers only to the quantum dots in Example 1, and does not include the quantum dots in Example 2 and Example 3. The quantum dots in Example 2 are only CDs-API-2, the quantum dots in Example 3 are only CDs-API-3, and the quantum dots in Comparative Example 1 are only CDs-API-4.

[0088] (I) Transmission electron microscope test

[0089] The shape and size of the materials were observed by transmission electron microscopy (TEM). The results showed that ChACDs and CDs-API were monodispersed, spherical with similar diameters and uniform size. High-resolution TEM images ( Figure 1 b upper right) shows that the lattice distance of CDs-API is 0.22nm and the crystal structure is consistent with graphene, revealing the typical graphite-like structure formed during the synthesis process.

[0090] (II) Dynamic light scattering test

[0091] The particle size of the nanomaterials was analyzed by dynamic light scattering (DLS), and the average size of ChACDs was found to be 2.35 nm, and the average size of CDs-API was 2.38 nm ( Figure 1 a inset, 1b inset). After modification of API, the shape and size of CDs did not change significantly.

[0092] (III) Fluorescence test

[0093] In addition, the synthesized ChACDs and CDs-API can emit dazzling blue fluorescence under 365nm excitation light, and after modification of API, the fluorescence of ChACDs solution is significantly enhanced ( Figure 1 c). This indicates that the presence of surface functional groups may affect the fluorescence properties of carbon quantum dots. Different functional groups can change the surface properties of carbon quantum dots, thereby affecting their fluorescence quantum yield; specific functional groups can also act as electron donors or acceptors, participate in the energy transfer process, and thus regulate the photoluminescence behavior of carbon quantum dots; by introducing specific functional groups through surface modification of carbon quantum dots, not only can the solubility of carbon quantum dots be changed, but also their fluorescence properties can be regulated, which may be of great significance for the development of fluorescent materials and biomarkers based on carbon quantum dots.

[0094] (IV) X-ray test

[0095] The elemental composition, functional group composition and surface structure of ChA CDs and CDs-API were further characterized. The elemental composition of ChACDs and CDs-API was analyzed by X-ray photoelectron spectroscopy (XPS). The results showed that ChACDs were mainly composed of C and O elements ( Figure 2 a). In the high-resolution XPS spectrum of the carbon 1s orbital, three characteristic peaks were observed. These peaks correspond to different carbon atom environments, suggesting the presence of different functional groups on the surface of carbon quantum dots, located at 284.8, 286, and 289 eV, respectively ( Figure 2 b), respectively, carbon-carbon bonds, carbon-oxygen bonds, and carbon-oxygen double bonds. This indicates that the surface functional groups of ChACDs are mainly composed of carbon atoms and abundant hydroxyl, carbonyl, and carboxyl groups.

[0096] (V) XPS test

[0097] Next, the charge conversion group API was modified onto the surface of ChACDs by the NHS / EDC method, and XPS confirmed the successful introduction of the N element ( Figure 2 c); In order to analyze the functional group composition of ChACDs and CDs-API and study the changes that occurred during the CDs-API reaction, the Fourier transform infrared spectra (FTIR) of ChACDs and CDs-API were further determined ( Figure 2 d), similar characteristic absorption peaks appeared in the two groups of samples. The existence of these characteristic peaks indicates that ChACDs and CDs-API have similar functional group compositions, such as the stretching vibrations of carbon-hydrogen bonds and carbon-oxygen bonds. In the absorption spectrum of CDs-API, new characteristic absorption peaks can be observed, such as 1545cm -1 The nitrogen-hydrogen bond at 1448 cm -1 The carbon-nitrogen bonds at the two positions are both amide bonds, which proves that the API is connected to the ChACDs surface via amide bonds, that is, the charge conversion group is successfully modified onto the ChACDs surface.

[0098] (VI) CDs-API acid response charge conversion ability test

[0099] The Zeta potential of ChACDs and CDs-API in a neutral environment (pH = 5.5) and an acidic environment (pH = 7.5) was measured and analyzed by dynamic light scattering (DLS); specifically, the following steps were performed: preparing 1 mg / mL ChACDs and CDs-API PBS solutions, respectively, and measuring the Zeta potential of the nanomaterials at pH = 5.5, 25°C and pH = 7.5, 25°C, respectively.

[0100] The results are as follows Figure 3As shown in the figure, in a neutral environment of pH = 7.5, both ChACDs and CDs-API are negatively charged, which to a certain extent avoids damage to healthy cells and tissues and enhances the biosafety of the material; in the acidic environment of bacterial biofilm (pH ≤ 5.5), the negative charge of ChACDs decreases, which may be attributed to its abundant surface groups: when the pH decreases, the H+ concentration in the solution increases and combines with the carboxyl groups on the surface of ChACDs, and the carboxyl groups change from -COO- in a neutral environment to -COOH, while the hydroxyl groups remain in the form of -OH; at this time, both the carboxyl group (-COOH) and the hydroxyl group (-OH) are not negatively charged, so as the pH decreases, the negative charge on the surface of ChACDs will also decrease to a certain extent; and CDs-API quickly reverses its charge to a positive charge (pKa < 6.8) due to the protonation of the imidazole group on the surface, and this charge reversal is not only conducive to the aggregation of the material around bacteria in the biofilm, but also gives ChACDs a strengthened bactericidal effect of positively charged materials.

[0101] (VII) CDs-API glutathionease activity test

[0102] The glutathione peroxidase detection kit (DTNB method) was used to detect the glutathione peroxidase catalytic activity of CDs-API (the new substance TNB generated by the reaction of DTNB and reduced GSH has a characteristic absorption peak at 412nm, and its absorbance is proportional to the GSH content, (see Figure 4 a); specifically comprising: first diluting CDs-API to 1 mg / mL with prepared PBS, adding DTNB solution (100 μM) and GSH solutions of different concentrations (1 mM, 2 mM, 3 mM, 4 mM) to the diluted solution, incubating at room temperature, and detecting the absorbance of the solution at 412 nm with an ELISA reader at different incubation times (0 min, 10 min, 20 min, 30 min, 40 min).

[0103] The results are as follows Figure 4 As shown in b, time-dependent and GSH concentration-dependent absorbance changes were observed at 412 nm ( Figure 4 b), indicating that GSH was effectively removed by CDs-API. Reduced GSH plays an important role in maintaining bacterial redox balance. GSH consumption can promote ROS accumulation in the biofilm environment. This type of GSH enzyme activity provides the material with effective bactericidal potential.

[0104] 3. In vitro antibacterial test of quantum dot CDs-API

[0105] In the following experiments, CDs-API refers only to the quantum dots in Example 1, excluding the quantum dots in Example 2 and Example 3. The quantum dots in Example 2 are only CDs-API-2, and the quantum dots in Example 3 are only CDs-API-2.

[0106] Take 5 μL of frozen glycerol culture of MRSA (methicillin-resistant Staphylococcus aureus) and PAE (penicillin-resistant enterococci) and inoculate it into a screw-capped tube containing 5 mL of LB liquid culture medium. The inoculated culture medium is placed in a constant temperature shaker and cultured at 37°C and 200 rpm. During the bacterial growth process, the absorbance value (OD) of the bacterial solution at a wavelength of 600 nm is regularly measured using a biological spectrophotometer. 600 ). When the absorbance value reaches 0.5, the amount of bacteria is about 10 8 CFU / mL, at this time the bacteria are in the logarithmic growth phase.

[0107] (I) In vitro antibacterial activity test

[0108] Take appropriate amount of MRSA and PAE strains and inoculate them into screw-capped tubes containing LB liquid medium, place them in a constant temperature shaker, and culture them at 37°C, 220rpm for 3 hours, at which time the bacteria are in the logarithmic growth phase. Dilute the bacterial solution with PBS (0.01M, pH 7.4) to a bacterial volume of about 10 6 CFU / mL; Use PBS to prepare ChACDs, CDs-API and CDs-API-4 into 1 mg / mL solutions respectively; Take 100 μL of PBS, ChACDs, CDs-API and CDs-API-4 solutions respectively and place them in a 96-well plate, add 100 μL of the above bacterial suspension to each well and mix, and incubate at room temperature for 20 minutes; After 20 minutes, take 100 μL of the mixed sample and inoculate it on the LB agar plate, use a spreader to evenly spread the sample on the surface of the agar plate to ensure that the bacteria are evenly distributed and let it stand for 10 minutes; Place the plate upside down and incubate it in a constant temperature incubator at 37°C for 18 hours; After 18 hours, take out the plate and take pictures to record the bacterial growth on each agar plate and count the number of colonies to evaluate the effect of the antimicrobial agent. In addition, 5 μL of the mixed system was inoculated into 5 mL of LB liquid medium in each well and cultured overnight on a constant temperature shaker at 37°C and 200 rpm. The optical density (OD) of the bacterial solution at 600 nm was measured using a spectrophotometer. 600 ).

[0109] The results showed that ChACDs and CDs-API had significant antibacterial effects against MRSA and PAE, and at the same concentration, the antibacterial effect of CDs-API was superior ( Figure 5a), while the blank control results of CDs-API-4 and PBS were similar, with colonies growing all over the plates, indicating that there was no antibacterial effect; at the same time, the OD of LB liquid culture medium under different treatments was measured by spectrophotometer 600 , the bacterial growth was evaluated by statistical analysis of the changes in absorbance values, and ChACDs and CDs-API obtained the same results as the plate experiment ( Figure 5 b and Figure 5 c); Similarly, CDs-API-4 and PBS blank controls showed dark purple and had higher absorbance, which was also the same as the results of the plate experiment.

[0110] (II) Determination of minimum inhibitory concentration

[0111] MRSA and PAE strains were cultured in LB liquid medium and grown in a 37°C constant temperature shaker to the logarithmic phase. The bacterial solution was diluted with PBS (0.01 M, pH 7.4) to a bacterial volume of about 10 6 CFU / mL.

[0112] ChACDs and CDs-API were prepared with PBS to different concentrations (the final concentrations were 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL and 1 mg / mL, respectively); 100 μL of PBS, ChACDs and CDs-API solutions were placed in a 96-well plate, 100 μL of the above bacterial suspension was added to each well and mixed, and incubated at room temperature for 20 min; after 20 min, the same operation as the in vitro antibacterial activity experiment was adopted, and the growth of bacteria in each group was observed on LB liquid culture medium and LB agar plate.

[0113] like Figure 6 The results showed that as the concentration of CDs-API increased, the colonies of MRSA and PAE decreased and the growth inhibition became more obvious, indicating that the antibacterial activity of CDs-API was concentration-dependent. The inhibition rate of CDs-API against MRSA and PAE reached 99% at a concentration of 1 mg / mL (Table 1).

[0114] Table 1 Inhibitory rate of different concentrations of CDs-API against MRSA and PAE

[0115]

[0116] In addition, the antibacterial rates of CDs-API-2 and CDs-API-3 at a concentration of 1 mg / mL were tested according to the above method, and the results are shown in Table 2 below.

[0117] Table 2 Inhibitory rate of CDs-API-2 and CDs-API-3 against MRSA and PAE

[0118]

[0119] (III) Broad-spectrum antibacterial activity test

[0120] Three strains of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) and ESBLs-producing K. pneumoniae were selected as models for evaluating the in vitro broad-spectrum antibacterial ability of CDs-API. Monoclonal colonies were picked from the agar plate and inoculated into a screw-capped tube containing LB liquid culture medium. The inoculated screw-capped tube was placed in a constant temperature shaker and incubated at 37°C and 200 rpm for 3 hours until the bacteria grew to the logarithmic phase (OD 600 =0.5); 100 μL of PBS (0.01M, pH 7.4), ChACDs and CDs-API (final concentration of 1 mg / mL) were taken in a 96-well plate, and 100 μL of the pre-prepared bacterial solution was added to the wells containing different solutions, and the 96-well plate was gently shaken to ensure uniform mixing; incubated at room temperature for 20 minutes to allow the materials to fully function. 10 μL of the mixed solution was taken from each well and evenly spread on the LB agar plate; the coated plate was inverted and placed in an incubator at 37°C for overnight incubation. The next day, the plate was taken out and the generated colonies were photographed and recorded.

[0121] The results are as follows Figure 7 As shown, both ChACDs and CDs-API inhibited the growth of these bacteria, but the antibacterial ability of ChACDs was weak and unstable at the same concentration, while CDs-API had a better and more stable broad-spectrum antibacterial ability.

[0122] (IV) CDs-API’s ability to destroy mature biofilms

[0123] Crystal violet (CV) dye was used to detect the degree of destruction of bacterial biofilm. First, a sterile 96-well plate was prepared to collect MRSA and PAE bacterial cultures in the logarithmic growth phase, ensuring that the bacterial count was approximately 10 8CFU / mL; then, take 200 μL of each of the above two bacterial solutions and add them to the corresponding wells of the pre-prepared sterile 96-well plate; place the inoculated 96-well plate in a 37°C constant temperature incubator for 36 hours to obtain a mature biofilm; then carefully aspirate the bacterial suspension in the well, and the light yellow paste (PAE is yellow-green) left is the bacterial biofilm, and gently rinse the remaining suspended bacteria and culture medium with PBS; then add PBS, ChA CDs, and CDs-API solution to the biofilm (the final concentration of ChA CDs and CDs-API is 1 mg / mL), gently shake to mix the liquid, and place it in a 37°C constant temperature incubator for incubation for 3 hours; after taking it out, gently aspirate the supernatant, add 200 μL of 0.1% CV dye to each well at room temperature, and incubate it in a light-proof environment for 15 minutes to ensure that the biofilm is fully stained. Then wash it three times with PBS to completely remove the unbound CV dye. Next, 200 μL of 95% ethanol solution was added to each well to dissolve and elute the CV dye fixed on the biofilm. Finally, the absorbance value of the eluate in each well was detected at a wavelength of 590 nm using a multifunctional microplate reader to evaluate the content of the biofilm in each group.

[0124] The results are as follows Figure 8 As shown, compared with the PBS control group, ChACDs only had a certain destructive effect on mature biofilms, while the CDs-API treatment group had a more excellent destructive effect on the mature biofilms of MRSA and PAE.

[0125] (V) Bacterial morphology observation

[0126] The surface morphology of bacteria treated with different methods was observed using scanning electron microscopy (SEM). PAE and MRSA (10 8 CFU / mL), and incubated with PBS (0.01M, pH 7.4), ChACDs (1mg / mL) or CDs-API (1mg / mL) for 20min. Then, the bacterial precipitate was obtained by low-speed centrifugation (5000r, 3min), the supernatant was gently aspirated, and the excess culture medium was removed by washing with PBS 1-2 times. The collected bacterial precipitate was resuspended in 2.5% glutaraldehyde solution and the bacteria were fixed at 4°C for 24h. Then, it was dehydrated at room temperature for 10min in different concentrations of ethanol (50%, 70%, 80%, 90%, 100%), vacuum dried, sprayed with gold, observed and photographed with SEM.

[0127] The results showed that the bacteria in the PBS control group were normal in shape, MRSA was nearly spherical, and PAE was rod-shaped. The bacterial surface was smooth and the structure was intact ( Fig. 9). After being treated with CDs-API, the surfaces of the two bacteria were deformed, the cell walls and cell membranes shrank, collapsed, or even ruptured, the cells were stacked and adhered, and even the cell contents flowed out (red arrows). This indicates that the antibacterial ability of CDs-API may be obtained by destroying the cell walls or cell membranes of bacteria.

[0128] (VI) Verification of bacterial membrane system damage

[0129] The fluorescent dye propidium iodide (PI) usually cannot penetrate the intact cell membrane. However, when the cell membrane is damaged, PI can enter the cell and bind to DNA, emitting red fluorescence. This feature can be used as an indicator of changes in bacterial membrane permeability. Prepare MRSA and PAE bacterial cultures (10 6 CFU / mL), 500 μL of each, 100 μL of PBS and 100 μL of CDs-API solution (final concentration of 1 mg / mL) were added respectively, and incubated at 37°C for 1 h and 2 h, and 50 μM PI fluorescent probe solution was added to measure the fluorescence intensity at an excitation wavelength of 535 nm.

[0130] Compared with the PBS control group, the fluorescence intensity of the MRSA and PAE bacterial suspensions co-incubated with CDs-API was significantly enhanced, and after 2 h of co-incubation, the fluorescence intensity of the bacterial suspension was further enhanced ( Fig.10 ), which indicates that CDs-API has a damaging effect on the bacterial cell membrane, resulting in changes in bacterial cell membrane permeability. PI enters the bacteria, binds to the DNA in the bacterial nucleoid structure, and releases red fluorescence; the longer the incubation time, the more severe the cell membrane damage caused by CDs-API, the more PI enters, and the stronger the fluorescence intensity. This result shows that CDs-API has a significant damaging effect on the bacterial cell membrane, causing changes in cell membrane permeability, and this damaging effect is time-dependent.

[0131] Using the charge-dependent aggregation release characteristics of diSC3(5), as an indicator of bacterial membrane potential changes, the potential changes of the bacterial cell membrane phospholipid bilayer were monitored. 6 CFU / mL), 800 μL of each, 60 μL of diSC3(5) (4 μM) and 60 μL of KCl (100 mM) were added and incubated at room temperature for 1 hour. 100 μL of PBS and 100 μL of LCDs-API solution (final concentration of 1 mg / mL) were added to the samples respectively. The samples were incubated for 1 hour and 2 hours at room temperature. The fluorescence intensity was measured at an excitation wavelength of 620 nm.

[0132] The results showed that the fluorescence intensity of diSC3(5) in the MRSA and PAE bacterial suspensions in the CDs-API group was significantly enhanced, and the fluorescence intensity after 2 h of co-incubation was greater than that after 1 h ( Fig.11 ), indicating that CDs-API can effectively damage the bacterial cell membrane and change the membrane potential of the bacterial cell membrane. With the increase of incubation time, the membrane damage is aggravated and the membrane potential change is increased, causing more diSC3(5) to be released into the solution.

[0133] The above two experiments prove that CDs-API can effectively destroy the bacterial cell membrane structure, causing a series of membrane damage such as changes in bacterial membrane permeability and membrane potential.

[0134] 7. Study on bacterial metabolism level

[0135] First, prepare MRSA and PAE bacterial suspensions in the logarithmic growth phase and dilute them with PBS to a bacterial volume of about 10 6 CFU / mL, mix the above two bacterial solutions with PBS, 1mg / mL ChACDs, and 1mg / mL CDs-API, respectively, and incubate at room temperature for 30 minutes to allow the materials to fully exert their effects. After incubation, collect the bacteria by centrifugation (5000rpm, 3min). Move to ice, use bacterial lysis solution to lyse the collected bacteria, and centrifuge again (12000g, 5min, 4℃) to collect the supernatant. Prepare a sterile 96-well plate, add 100μL ATP working solution to each well, place it at room temperature for 5min, then add 100μCDs-API solution (1mg / mL) and mix gently, and finally use a multifunctional microplate reader to detect the chemiluminescence value of each well.

[0136] The results showed that compared with the PBS control group, the ATP content in the CDs-API treatment group decreased significantly ( Fig.12 ), indicating that the metabolic activity of bacteria decreased or even stagnated. This decrease in metabolic level can be regarded as a manifestation of bacterial death. It can be concluded that CDs-API can significantly affect the metabolic level of bacteria, causing the metabolic activity of bacteria to decrease or even stop, which may be one of the reasons for bacterial death.

[0137] (VIII) ROS signal detection

[0138] The singlet oxygen fluorescence probe ABDA (9,10-anthracenediyl-bis(methylene)dicarboxylic acid) was used as an indicator to monitor the decomposition of ABDA by the decrease in absorbance at 378 nm, thereby determining the content of ROS in the system. 50 μL of ABDA solution (500 mM) was mixed with ①10 8 CFU / mL MRSA, 10 8 CFU / mL PAE, 10 8CFU / mLMRSA+1mg / mL ChACDs, 10 8 CFU / mL PAE+1mg / mL ChA CDs; ②10 8 CFU / mL MRSA, 10 8 CFU / mL PAE, 10 8 CFU / mLMRSA+1mg / mL CDs-API, 10 8 CFU / mLPAE+1mg / mL CDs-API were mixed and incubated at room temperature, and the absorbance of the mixed system at 378nm at different reaction times (10min, 20min, 30min, 40min) was detected by an enzyme reader.

[0139] like Fig.13 The results showed that the absorbance of ABDA did not decrease significantly when incubated with bacteria, indicating that bacteria do not produce a large amount of ROS during normal growth; however, when ChACDs and CDs-API were incubated with bacteria, the absorbance decreased significantly, and the absorbance continued to decrease with the increase of incubation time, proving that both ChACDs and CDs-API can promote the accumulation of bacterial ROS, and ROS continues to increase with time.

[0140] (IX) In vitro biocompatibility of CDs-API

[0141] Human umbilical vein endothelial cells (HUVEC) were selected as the research object for in vitro cytotoxicity study. First, HUVEC cells were cultured at 5×10 3 The cells were inoculated at a density of 10 cells / well in a 96-well plate, and the cells were incubated with a series of concentrations of CDs-API (1mg / mL, 3mg / mL, 5mg / mL, 7mg / mL, 10mg / mL) for 48 hours, and the cell survival rate was determined by the MTT method. 10μL of methyl thiazolyltetrazolium (MTT, 0.5mg / mL, pH=7.4) was added to each well, and the culture was terminated after incubation for 4 hours. The supernatant in the well was gently aspirated, and 100μLDMSO was added to each well. The shaker was shaken for 10 minutes to fully dissolve the mitochondria of the living cells and reduce MTT to blue-purple crystalline formazan. Finally, the absorbance value of the mixed system in each well at 490nm was detected with a multifunctional microplate reader, and the results were recorded.

[0142] The results are as follows Fig.14 Even at a high concentration of 10 mg / mL, the cell survival rate was almost unaffected, indicating that CDs-API has almost no cytotoxicity in vitro and can be used for in vivo anti-infection treatment experiments.

[0143] IV. Quantum Dots In Vivo Test

[0144] In the following experiments, CDs-API refers only to the quantum dots in Example 1, excluding the quantum dots in Example 2 and Example 3. The quantum dots in Example 2 are only CDs-API-2, and the quantum dots in Example 3 are only CDs-API-2.

[0145] The animals used in this experiment have been approved by the Experimental Animal Welfare Ethics Review Committee of Chongqing Medical University. Eighteen 6-week-old female BALB / c mice were selected and subjected to a one-week adaptive feeding for skin suppurative infection modeling. First, the healthy mice were randomly divided into two groups: MRSA infection group and PAE infection group, and then the two groups were divided into three subgroups (n=3), namely: PBS blank control group, ChACDs treatment group and CDs-API treatment group. The back of the mouse was depilated, and a circle with a diameter of about 9 mm was marked at a fixed position with a hole punch. Under ether gas anesthesia, surgical scissors were used to cut off the marked circular skin area on the back of the mouse, and 100 μL MRSA bacterial solution and PAE bacterial solution (10 8 CFU / mL) was applied to the open wound site after the skin was cut off. After 24 hours of cage rearing, obvious pus could be observed in the wound site, proving that the skin suppurative infection model was successfully established.

[0146] (I) Biocompatibility of CDs-API in vivo

[0147] After successfully establishing a mouse back wound infection model, CDs and CDs-API were treated according to different groups, and the weight changes of the mice were continuously recorded; after the treatment, the mouse organs were surgically removed for H&E staining to evaluate the toxicity of the materials to the mouse organs. The results showed that during the 12-day treatment, the weight of the mice did not decrease significantly, and maintained a steady increase over time ( Fig.15 ); HE staining results of major organs showed that compared with the PBS control group, the morphological structures of various organs in the CDs and CDs-API treatment groups were normal, with no obvious toxic damage or histological changes ( Fig.16 These results prove that CDs-API has no obvious toxicity to mouse organs, has high biocompatibility, does not cause damage to the body when treating bacterial infections, and is highly safe.

[0148] (II) CDs-API in vivo anti-infection treatment trial

[0149] PBS (0.01M, pH 7.4), ChACDs (1 mg / mL) and CDs-API (1 mg / mL) were administered directly into the wound of each mouse at a dose of 50 μL. During the entire treatment period, different treatment drugs were administered at a fixed time every day, and the changes in wound size were recorded (see Fig.17 ). The weight of the mice was recorded every other day; on the 12th day, the mice were sacrificed, the pus from the infection site was collected, diluted with PBS and inoculated into LB liquid culture medium and LB agar plate, incubated in a constant temperature incubator at 37°C for 18 hours, and the growth of the colonies was recorded by taking pictures. In addition, the wound tissue and surrounding skin on the back of the mice were obtained surgically, and the main organs (heart, liver, spleen, lung, and kidney) were removed by dissection. The tissues and organs were fixed with 4% paraformaldehyde solution for 48 hours. The fixed tissue was dehydrated and paraffin-embedded to prepare tissue sections with a thickness of 4-6μm. The prepared tissue sections were stained with hematoxylin and eosin (H&E), placed in a slice panoramic scanning system for photography, and the structure and pathological conditions of the tissues were evaluated.

[0150] On the 6th day of treatment, it was observed that the wounds of the PBS control group mice in the MRSA and PAE infection groups still had obvious edema and ulceration, while the wounds of the CDs and CDs-API treatment groups were significantly reduced and gradually healed with scabs ( Fig.18 a, 18b, 19a, 19b); at this stage, the pus from the wounds of each group of mice was collected and inoculated into LB liquid culture medium. After incubation for 18 hours, it was observed that the bacterial liquid in the PBS control group was obviously turbid, while the suspensions in the ChACDs group and the CDs-API group were clear and transparent ( Fig.18 b, Fig.19 b), which indicates that bacterial growth has been inhibited on the 6th day of treatment. As the treatment continued, the wounds of mice in the PBS control group repeatedly ruptured, proving that bacteria in the wounds were still proliferating; on the contrary, the wounds of mice in the ChACDs and CDs-API treatment groups gradually scabbed and healed. At the end of the treatment, the wounds of mice in the CDs-API group were almost completely healed, and the healing speed was faster than that of the ChACDs treatment group, indicating that both ChACDs and CDs-API have certain antibacterial ability in vivo, and CDs-API has a better effect than ChACDs.

[0151] After 12 days of treatment, the mice were sacrificed, and the bacterial suspension from the infected wounds of the mice was obtained and cultured in LB solid medium. After incubation for 18 hours, the colonies on the solid medium were counted to evaluate the residual amount of bacteria in the infected area. Fig.18As shown in Figs. 1a and 1b, compared with the control group, there was almost no single colony formation in the bacterial solution plating of the wound infection site of the mice in the ChACDs and CDs-API treatment groups, indicating that the number of bacteria in the wound site was negligible, which was consistent with the results of the in vitro antibacterial ability test, further proving the antibacterial ability of the material. At the same time, the tissues of the wound infection site were collected for H&E staining to evaluate the degree of infection of the wound. It can be seen that there is still a large amount of inflammatory cell infiltration in the wound site of the mice in the PBS control group (red arrow), and the inflammatory cell infiltration in the infected tissue of the ChACDs and CDs-API treatment groups is significantly reduced ( Fig.18 c, 19c), demonstrating that bacterial infection was fully inhibited.

[0152] The above results show that both ChACDs and CDs-API have the ability to inhibit the proliferation of MRSA and PAE in vivo, reduce inflammation by killing bacteria, and promote skin recovery at the wound site of mice. In contrast, CDs-API can kill bacteria at the infected site in a shorter time and has a better therapeutic effect.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A method for preparing environmentally responsive charge conversion carbon quantum dots, characterized in that: include: (1) Chlorogenic acid is made into quantum dots to obtain ChACDs; (2) ChACDs are modified with imidazole compounds to obtain environmentally responsive charge-converting carbon quantum dots.

2. The preparation method according to claim 1, characterized in that: Imidazole compounds are n is an integer and 1≤n≤5.

3. The preparation method according to claim 2, characterized in that: The imidazole compound is 1-(3-aminopropyl)imidazole.

4. The preparation method according to claim 2 or 3, characterized in that: The mass / volume of ChACDs and imidazole compounds was (3-6) mg / mL.

5. The preparation method according to any one of claims 1 to 3, characterized in that: The preparation method meets one or more combinations of the following conditions: (a) In step (1), ChACDs are prepared from chlorogenic acid by a hydrothermal synthesis method; (b) In step (2), the imidazole compound is modified to ChACDs by the EDC / NHS method.

6. The preparation method according to claim 5, characterized in that: Step (1) comprises: placing the ChA aqueous solution in a high pressure reactor, and then heating it at 220° C.-240° C. to obtain a ChACDs aqueous solution; Step (2) comprises: (i) mixing EDC, NHS and 2-morpholineethanesulfonic acid to obtain a reactant; (ii) mixing the reactant with an imidazole compound, adjusting the pH to 7-9, and reacting to obtain environmentally responsive charge conversion carbon quantum dots.

7. Environmentally responsive charge conversion carbon quantum dots prepared by the preparation method according to any one of claims 1 to 6.

8. Any of the following substances: (i) A composition comprising the environmentally responsive charge conversion carbon quantum dots according to claim 7; the composition is used for antibacterial, inhibiting the growth of drug-resistant bacteria, treating skin suppurative infections or promoting wound healing; (ii) a preparation comprising the environmentally responsive charge conversion carbon quantum dots according to claim 7 or the composition in (i); and carrier.

9. Use of the environmentally responsive charge-converting carbon quantum dots described in claim 7 in antibacterial applications.

10. The use according to claim 9, characterized in that: Applications include: Environmentally responsive charge-converting carbon quantum dots for antibiotic-resistant bacteria.

11. The use according to claim 10, characterized in that: Antibiotic-resistant bacteria include one or more of methicillin-resistant Staphylococcus aureus, penicillin-resistant Enterococci, vancomycin-resistant Enterococci, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, or pan-resistant Acinetobacter.

12. The use according to any one of claims 9 to 11, characterized in that Applications include: use of environmentally responsive charge-converting carbon quantum dots in the preparation of compositions or preparations, the compositions or preparations being used to destroy bacterial membrane systems, reduce bacterial metabolic levels, or promote bacterial ROS accumulation.

13. The use according to any one of claims 9 to 11, characterized in that Applications include: use of environmentally responsive charge-converting carbon quantum dots in the preparation of pharmaceutical compositions or pharmaceutical preparations, and the pharmaceutical compositions or pharmaceutical preparations are used to treat suppurative skin infections and / or promote wound healing.

Images